Evolution of human H3N2 influenza virus receptor specificity has substantially expanded the receptor-binding domain site

Summary: Hemagglutinins (HAs) from human influenza viruses descend from avian progenitors that bind α2–3-linked sialosides, and must adapt to glycans with α2–6-linked sialic acids on human airway cells to transmit within the human population. Since their introduction during the 1968 pandemic, H3N2 viruses have evolved over the past five decades to preferentially recognize α2–6-sialoside receptors that are elongated through addition of poly-LacNAc. Using STD-NMR, X-ray crystallography, and solid-phase glycan microarrays, we show that more recent H3N2 viruses now make increasingly complex interactions with elongated receptors, while continuously selecting for strains maintaining this phenotype. This change is accompanied by an extension of the traditional receptor binding site to include residues in key antigenic sites on the surface of HA trimers. These results help explain the propensity for selection of antigenic variants, leading to vaccine mismatching, when H3N2 viruses are propagated in chicken eggs or cells that do not contain such receptors.


Figure S2. Structural overview of influenza HA
The HA glycoprotein of influenza (H3N2) viruses is a two-domain homotrimer, represented here as molecules A, B, & C shown in silver, pink, and blue, respectively; featuring HA head and stem domains.The canonical receptor binding site (RBS; shown highlighted via a black dashed box) is located in the upper part of the head domain and (shown inset) is made up of three well-characterized structural motifs, the 130/140 loop region, the 190 helix, and 220 loop.The 150 loop, located to the side of the RBS, is traditionally associated with antigenicity and a target for antibody binding.However, highly conserved Trp-153 at the base of the 150 loop is a key residue for sialic acid receptor binding.The example HA structure shown here is of A/Hong Kong/01/68 H3N2 (HK/68; PDB ID 6TZB).Figure assembled using PyMOL (Schrodinger, LLC).

Figure S3. H3-receptor complex structures
Panels A -K show candidate receptor terminal fragments, either α2-6-sialyl-LacNAc (6SLN; panels C and F), LSTc (panel H), or α2-6-sialyl-(LacNAc)2 (6SLN2; all other panels), bound to representative H3 influenza glycoproteins covering approximately 3 -10 yearly intervals over the last five decades.All panels represent identical views of the respective H3 complexes and feature two images of the RBS shown at 90° rotations around the y-axis relative to one another.Key receptor-binding side chains of residues 98, 135 -137, 145, 156, 159, 183, 190, 193, 222, 225, & 225, are shown in all panels (see enlarged versions of panels A & K in Figure 5 for labels); key highly-coordinate water molecules indicating indirect receptor-binding interactions are shown as red spheres, while H-bonds are depicted as black dashed lines.Panels A, F, G, & H show previously published structures (PDB IDs 6TZB, 6BKR, 6AOV, & 5XRS, respectively), while other panels represent novel structures as part of this work.Panels assembled using PyMOL (Schrodinger, LLC).

Figure S4. Nextstrain influenza clade phylogeny
Phylogeny trees of human H3N2 viruses over the last 1.5 decades produced by Nextstrain (https://nextstrain.org) and colored by (A) clade and (B) residue identity at HA position 159.As shown, following evolution from F159 around 2013 onwards, Y159 or S159 diverge and are found exclusively in either clades 3C.2a-descended or 3C.3a-descended strains, respectively.n.b.Bold "X" markings depict vaccine strains selected within the different clades over time.The color scale within individual columns (array datasets) are independently scaled to the most intense RFU within that group.Blue arrows highlight short receptors within a given group containing only 2 LacNAc repeats.Columns depict data collected from various different Sialoside array versions, including V1, V3, V4, & V5.Receptor structures corresponding to glycan numbers for all array versions can be found in Supplementary Data S1.Grey bars illustrate that a given receptor structure was absent in the array version on which a particular dataset was collected.Individual array plots for all mutants, including binders shown here and non-binders, are illustrated in Supplementary Data S2.The color scale within individual columns (array datasets) are independently scaled to the most intense RFU within that group.Blue arrows highlight short receptors within a given group containing only 2 LacNAc repeats.Columns depict data collected from various different Sialoside array versions, including V3, V4, & V5.Receptor structures corresponding to glycan numbers for all array versions can be found in Supplementary Data S1.Grey bars illustrate that a given receptor structure was absent in the array version on which a particular dataset was collected.Individual array plots for all mutants are illustrated in Supplementary Data S2.

Figure S5 .
Figure S5.Structural comparison of receptor binding in clade 3C.2a and 3C.3a H3 HAs Close up structural views of the 150-loop in 6SLN2-receptor bound H3 representatives from clades 3C.2a (A/Texas/73/2017, Tex17; pink) and 3C.3a (A/Ecuador/1374/2016, Ecu16; teal).(A) Reduced steric interference from the smaller S159 side chain in 3C.3a HAs allows greater freedom at the reducing end of the receptor molecule, allowing the terminal GlcNAc sugar to rotate freely compared to the relatively fixed position in 3C.2a and earlier H3 structures.(B) & (C) Surface comparisons showing the substantial additional steric bulk imposed by Y/F159 side chains, compared to S159.Larger arylcontaining side chains push the reducing-end GlcNAc out from the HA surface, while substantial interactions with sugars below this position hold the glycan chain close to the surface.Thus longer saccharide chains with potentially greater flexibility are required to navigate this added surface complexity.Panels assembled using Pymol (Schrodinger, LLC).

Figure S6 .
Figure S6.Glycan microarray analysis of Vic/11 to HK/68 RBS reversion mutants Heatmap representation of glycan microarray data comparing Vic/11 WT to RBS-proximal variants where amino acid species have been reversed to their original identity in the post-pandemic HK/68 strain.Only variants giving rise to detectable binding are shown.WT Vic/11 (far left column) maintains a characteristic pattern of strong binding to extended receptors with little or no interactions with shorter or α2-3-terminal glycans.All variants, as far as a 13-mutant HA, result in either no binding (not shown in this figure) or limited alteration of the WT Vic/11 specificity pattern (compare columns moving left to right).The final triple mutants shown on the right of the panel reveal the beginnings of a non-binding phenotype, with native specificity almost entirely lost and widespread non-specific binding to almost all receptors visible.

Figure S7 .
Figure S7.Glycan microarray analysis of variants restoring receptor binding in mutated Vic/11 backgroundsAddition of two variants, F159Y (A) and N225D (B), that evolved immediately before K160T in clade 3C.2a H3s restore receptor binding in a Vic/11-K160T background that alone inhibits receptor engagement through creation of a novel glycan at position N158.While both F159Y and N225D promote positive binding compared to K160T alone, the K160T-N225D double variant in panel (B) clearly shows the most native-like receptor specificity, indicating this variant has the dominant positive effect.Panel (C) shows a glycan microarray heatmap of WT Vic/11 compared to RBS-proximal variants reverted to respective HK/68 amino acid species without and with N225D.Latter versions (righthand 3 columns) all show stronger and far more comparable binding specificity to WT, while variants without N225D show substantially disrupted, non-specific binding.